Naked antisense oligonucleotides access the cytosol during early endocytic trafficking

Abstract

Phosphorothioate-modified antisense oligonucleotides (ASOs) enter cells, but their site of productive escape remains uncertain. We combined live-cell imaging and ratiometric pH measurements to follow a Malat1-targeting ASO internalized without a delivery vehicle. In U2OS cells, less than 4% of internalized ASO reached the nucleus under conditions that reduced Malat1 RNA by >90%. Most ASO accumulated in late endosomes, endolysosomes and lysosomes as foci and broadly distributed luminal signal. These compartments underwent neutralization without detectable membrane rupture or galectin-3 recruitment. ASO foci showed constrained motion, consistent with association with endolysosomal constituents. L-leucyl-L-leucine methyl ester-induced membrane damage or Calcium dependent lysosome exocytosis released dextran while retaining luminal ASO. Loss of the stress-granule scaffolding proteins G3BP1/2 modestly enhanced ASO activity without detectable endolysosomal damage. Tantalosin, a small molecule that perturbs endosomal recycling, redistributed the ESCRT-III protein IST1 to endosomes. It enhanced early escape of SYTOX Green and ASOs, detected by nuclear accumulation, and increased Malat1 knockdown, with cell-type-dependent responses. Tantalosin did not detectably increase nuclear ASO accumulation after sequestration in late compartments. These findings place productive ASO escape early in endocytic trafficking, most likely in early or recycling endosomes, before sequestration in largely nonproductive late compartments.